Ft-0554a substances and process for the preparation thereof

ABSTRACT

The present invention relates to obtain novel FT-0554A substance useful for infectious disease of parasite, especially helminth and more particularly relates to the novel FT-0554A substance represented by the following formula [I]                   
     or the tautomer thereof represented by the formula [II]                   
     obtained by treating FT-0554 substance represented by the formula [II]                   
     under alkaline condition.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

This invention relates to novel FT-0554A substance useful for treatmentfor infection of parasite, especially helminth, and its production.

(2) Description of the Related Art

Parasitosis is reducing as a result of improvement in sanitaryconditions and progress of anthelmintics. Recently, however, the importparasitosis, zoonotic parasitosis, opportunistic parasitosis andparasitosis originated from perishable foods are prevailing and becomecrucial problems. Further the parasitosis produces large economicalburdens in the stock-farming and agriculture. For infection of helminthin the parasite, at present, avermectins, mebendazole, praziquantel, andothers are used for treatment of helminth.

SUMMARY OF THE INVENTION

Anthelmintics used at present, such as avermectins, mebendazole andpraziquantel, are not always sufficient for satisfactory in efficacy andtoxicity, in addition, helminth which acquired resistance to theconventional anthelmintics is increasing, consequently novel drugs arestrongly required.

We have studied NADH-fumarate reductase, which was one of the promisingtargets against anthelmintics, in the electron transport system of thehelminth, and explored for the inhibitor of the NADH-fumarate reductasefrom the microbial culture. As a result, we have found that FT-0554substance produced by a strain belonging, to genus Aspergillus,Aspergillus niger had NADH-fumarate reductase inhibitory activity, andhad made international patent application (international publication no.WO99/24439).

The above described Aspergillus niger is described in Boris Schilling etal., “Amine oxidases from Aspergillus niger:identification of a novelflavin-dependent enzyme”, Biochimica et Biophysica Acta Vol. 1243 (1995)p. 529-537, and Yoshiharu Inoue et al., “Metabolism of 2-oxoaldehyde inmold. Purification and characterization of two methylglyoxal reductasesfrom Aspergillus niger”, Eur. J. Biochem Vol. 171 (1988) p. 213-218.

As a result of our continued studies, we have found that novel FT-0554Asubstance derived from FT-0554 substance had also equivalent level ofinhibitory activity of NADH-fumarate reductase, and completed thepresent invention.

Namely, the present invention relates to FT-0554A substance representedby the formula [I]:

or its tautomer of the formula [II]:

Further, the present invention relates to FT-0554A substance having thefollowing physicochemical properties.

(1) Nature: white powder or amorphous

(2) Molecular weight: 383.2201 (M+Na, high resolution fast atombombardment mass spectrometry)

(3) Molecular formula: C₂₂H₃₂O₄

(4) Melting point: 101-103° C.

(5) Specific rotation: [α]_(D) ²⁶=+4.6° (c=1, chloroform)

(6) IR absorption maximum (KBr Tab): As shown in FIG. 1, maximumabsorption at 3375, 2960, 2926, 1755, 1738, 1660, 1456, 1261, 1074,1024, and 800 cm⁻¹

(7) Solubility in solvent: soluble in chloroform and ethyl acetate;insoluble in water and n-hexane,

(8) Color reaction: positive for phosphomolybdic acid reagent.

Further, the present invention relates to a process for production ofFT-0554A substance of the formula [I]

or its tautomer of the formula [II]

wherein FT-0554 substance represented by the formula [III]

is treated by alkali.

The present invention further relates to NADH-fumarate reductaseinhibitor comprising FT-0554A substance as an active ingredient.

Furthermore, the present invention relates to antihelminthic agentcomprising FT-0554A substance as an active ingredient.

Detailes physicochemical properties of FT-0554A substance of the presentinvention are as follows.

(1) Nature: white powder or amorphous

(2) Molecular weight: 383.2201 (M÷Na, high resolution fast atombombardment mass spectrometry)

(3) Molecular formula: C₂₂H₃₂O₄

(4) Melting point: 101-103° C.

(5) Specific rotation: [α]_(D) ²⁶=+4.60° (c=1, chloroform)

(6) IR absorption maximum (KBr Tab): As shown in FIG. 1, maximumabsorption at 3375, 2960, 2926, 1755, 1738, 1660, 1456, 1261, 1074,1024, and 800 cm⁻¹

(7) ¹H-proton Nuclear Magnetic Resonance spectrum: chemical shift ofFT-0554A substance [I] in deuteroacetone (ppm) is as follows.

s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad,H: number of proton, J: coupling constant (Hz)

 6.17 s (1H, H-3), 6.25 dd (1H, J=10.5, 15.1, H-7), 6.18 dd (1H, J=10.7,15.1, H-14), 6.02 dd (1H, J=10.5, 15.4, H-8), 5.74 d (1H, J=10.7, H-13),5.61 dd (1H, J=7.2, 15.4, H-9), 5.58 dd (1H, J=7.2, 15.1, H-6), 5.39 dd(1H, J=8.0, 15.1, H-15), 4.32 br.d (1H, J=4.0, 5-OH), 4.08 m (1H, H-5),2.40 m (1H, H-10), 2.05 m (1H, Ha-11), 2.01 m (1H, H-16), 1.93 m (1H,Hb-11), 1.67 s (3H, 12-CH₃), 1.32 s (3H, 4-CH₃), 1.25 m (2H, H₂-17),0.93 d (3H, J=6.6, 16-CH₃), 0.91 d (3H, J=6.9, 10-CH₃), 0.80 t (3H,J=7.4, H₃-18)

(8) ¹H-proton Nuclear Magnetic Resonance Spectrum: chemical shift ofFT-0554A substance [II] in deuteroacetone (ppm) is as follows.

s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad,H: number of proton, J: coupling constant (Hz)

 6.34 dd (1H, J=10.5, 15.1, H-7), 6.18 dd (1H, J=10.7, 15.1, H-14), 6.05dd (1H, J=10.5, 15.4, H-8), 5.74 d (1H, J=10.7, H-13), 5.67 dd (1H,J=7.2, 15.4, H-9), 5.61 dd (1H, J=7.2, 15.1, H-6), 5.39 dd (1H, J=8.0,15.1, H-15), 5.11 br.d (1H, J=3.6, 5-OH), 4.22 m (1H, H-5), 2.88 d (1H,J=18.7, Ha-3), 2.55 d (1H, J=18.7, Hb-3), 2.40 m (1H, H-10), 2.05 m (1H,Ha-11), 2.01 m (1H, H-16), 1.93 m (1H, Hb-11) 1.67 s (3H, 12-CH₃), 1.48s (3H, 4-CH₃), 1.25 m (2H, H₂-17), 0.93 d (3H, J=6.6, 16-CH₃), 0.91 d(3H, J=6.9, 10-CH₃), 0.80 t (3H, J=7.4, H₃-18)

(9) ¹³C-Nuclear Magnetic Resonance spectrum: chemical shift of FT-0554Asubstance [I] in deuteroacetone (ppm) is as follows.

s: singlet, d; doublet, t: triplet, q: quartet

 169.0 s (C-1), 141.4 d (C-9), 138.8 d (C-15), 134.74 s (C-12), 133.8 d(C-7), 130.0 d (C-6), 128.6 d (C-8), 127.7 d (C-13), 125.9 d (C-14),121.5 d (C-3), 86.2 s (C-4), 76.6 d (C-5), 48.1 t (C-11), 39.3 d (C-16),35.6 d (C-10), 30.4 t (C-17), 21.3 q (4-CH₃), 20.5 q (16-CH₃), 20.1 q(10-CH₃), 16.5 q (12-CH₃), 12.0 q (C-18), C2 cannot be identified.

(10) ¹³C-Nuclear Magnetic Resonance spectrum: chemical shift of FT-0554Asubstance [II] in deuteroacetone (ppm) is as follows.

s: singlet, d: doublet, t: triplet, q: quartet

 193.7 s (C-2), 161.5 s (C-1), 142.4 d (C-9), 138.9 d (C-15), 135.0 d(C-7), 134.66 s (C-12), 128.7 d (C-6), 128.3 d (C-8), 127.6 d (C-13)125.9 d (C-14), 84.9 s (C-4), 77.0 d (C-5), 48.2 t (C-11), 40.6 d (C-3),39.3 d (C-16), 35.5 d (C-10), 30.4 t (C-17), 23.3 q (4-CH₃), 20.5 q(16-CH₃), 20.0 q (10-CH₃), 16.5 q (12-CH₃), 12.0 q (C-18)

(11) Solubility in solvent: soluble in chloroform and ethyl acetate;hardly soluble in water and n hexane

(12) Color reaction: positive for phosphomolybdic acid reagent.

As a result of detailed examination of physico-chemical properties andspectra data of FT-0554A substance, FT-0554A substance is determined asthe following chemical structure [I]:

or its tautomer of the formula [II]:

As shown in the above, physico-chemical properties of FT-0554A substanceare described in detail, however no compound having identical propertieshas been reported. Consequently, FT-0554A substance is defined as novelsubstance.

Next, the production of FT-0554A substance of the present invention fromFT-0554 substance is explained concretely.

A microorganism having FT-0554 substance producing activity belonging toAspergillus, Aspergillus niger FT-0554 FERM BP-6443, disclosed in theinternational publication no. WO99/24439 is cultured in the medium, toaccumulate FT-0554 substance in the cultured medium and to isolateFT-0554 substance represented by the following formula [III] from thecultured medium.

The thus obtained FT-0554 substance is treated under alkaline conditionto produce FT-0554A substance of the present invention.

Namely, FT-0554 substance is dissolved in the conventional organicsolvent, for example, alcohol such as methanol, ethanol, etc., ethersuch as diethyl ether, dimethyl sulfoxide and chloroform, and basiccompound such as potassium carbonate, sodium carbonate, potassiumhydrogen carbonate, sodium hydrogen carbonate, sodium methoxide, sodiumhydride, sodium hydroxide, potassium hydroxide and anmmonium is added togenerate FT-0554A substance.

Purification and isolation of the substance from the reaction mixturecan be performed by the conventional methods used for purification ofthe common lipophilic organic compound, for example, extraction withwater immiscible organic solvent such as ethyl acetate, variouschromatographic methods such as absorption chromatography,gel-filtration chromatography, scratching from thin layerchromatography, centrifugal counter-current chromatography, and highperformance liquid chromatography, and precipitation from slightlysoluble solvent, with combination thereof or repeating these process, asa result, purified FT-0554A substance can be obtained.

NADH-fumarate reductase inhibitory activity of FT-0554A substance of thepresent invention is explained as follows.

Muscles of Ascaris suum were homogenized in 120 mM sodium phosphatesolution (pH 7.0) and centrifuged at 3000×g for 10 minutes to collectthe supernatant solution. The supernatant was further centrifuged at10000×g for 20 minutes to collect the precipitate. The precipitate wassuspended in 120 mM sodium phosphate solution (pH 7.0) to obtainmitochondorial fraction.

After 10 μl of test sample dissolved in 50% dimethyl sulfoxide solutionwas added into 96 wells microplate, 80 μl of 120 mM sodium phosphatesolution (pH 7.0) containing 0.35 mM NADH, 7.2 mM disodium fumarate and18 mg/ml bovine serum albumin was added thereto, and pre-incubated inthe microplate reader ELX808 (Bio-Teck Industries Co.) at 37° C. for 5minutes. Mithochondrial fraction of Ascaris suum 10 μl (protein content0.3 mg) was added therein and incubated at 37° C. for 10 minutes.Absorption of NADH at 340 nm was measured every 15 seconds.

As a result of quantitative measurement of NADH-fumarate reductaseactivity shown by decrease in the slope of absorbancy at 340 nm, 50%inhibition of NADH-fumarate reductase activity were obtained at 0.4 μMof FT-0554A substance. Consequently, FT-0554A substance can be expectedto use as drug for treatment or prevention of helminthiasis.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows IR spectrum of FT-0554A substance of the present invention(KBr Tab).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

The following example illustrates the present invention, but is notconstrued to limit the invention.

EXAMPLE

FT-0554A substance 10.0 mg was dissolved in methanol 280 μl, addedcalcium carbonate 2.0 mg therein and stirred. After kept at roomtemperature for 30 minutes, the reaction mixture was diluted by addingethyl acetate, added saturated sodium chloride and treated by theextraction operation. Organic layer was separated, dried by adding,anhydrous sodium sulfate, and concentrated in vacuo to obtain FT-0554Asubstance 9.3 mg, as white powder.

EFFECT OF THE INVENTION

As explained hereinabove, the present invention provides FT-0554Asubstance or the process for production of FT-0554A substance fromFT-0554 substance, and the thus obtained FT-0554A substance is expectedto be a satisfactory and useful medicament for treatment of parasitosis.

What is claimed is:
 1. A compound represented by formula [I]

or the tautomer thereof of formula [II]


2. A composition comprising a compound according to claim 1 having thefollowing physiochemical properties: (1) Nature: white powder oramorphous (2) Molecular weight: 383.2201 (M÷Na, high resolution fastatom bombardment mass spectrometry) (3) Molecular formula: C₂₂H₃₂O₄ (4)Melting point: 101-103° C. (5) Specific rotation: [α]_(n) ²⁶=+4.6° (c=1,chloroform) (6) IR absorption maximum (KBr Tab): As shown in FIG. 1,maximum absorption at 3375, 2960, 2926, 1755, 1738, 1660, 1456, 1261,1074, 1024, and 800 cm⁻¹ (7) solubility in solvent: soluble inchloroform and ethyl acetate, hardly soluble in water and n-hexane, and(8) Color reaction: positive for phosphomolybdic acid reagent, and aninert carrier.
 3. A process for production of a composition comprising acompound of formula [I]

or the tautomer thereof represented by formula [II]

comprising creating a compound represented by formula [III]

under alkaline condition.
 4. A method of inhibiting NADH-fumaratereductase in vitro comprising administering to a mitochondria suspensionan NADH-fumarate reductase inhibiting effective amount of a compositioncontaining a compound of formula [I]

or the tautomer thereof of formula [II]


5. A method of treating helminthiasis in a human or an animal or a humanor animal at risk of contracting helminthiasis, comprising administeringan effective amount of a composition to said human or animal and whereinsaid composition contains a compound of formula [I]

or the tautomer thereof of formula [II]


6. A method for producing a composition comprising a compound of formula[I]

or the tautomer thereof of formula [II]

said method comprising culturing in medium microorganism Aspergillusniger FT-0554 which produces a compound represented by formula [III]

isolating the compound represented by formula [III] from the culturedmedium, and treating the compound under alkaline conditions to produce acompound of formula [I]

or the tautomer thereof of formula [II]

and wherein compound I or compound II are added to said composition.